Enzymes find extensive applicability in diverse areas such as food processing, enantioselective organic synthesis, production of pharmaceuticals, clinical diagnosis/treatment, extracorporeal affinity chromatography, waste management, environmental analysis/pollution control and biosensors. As industrial catalysts, they offer a number of advantages over conventional chemical catalysts due to their high catalytic activity, substrate specificity, the mild conditions involved in their use, minimal by-product formation and no environmental pollution risk. However the two main disadvantages relating to their utility are their instability and the economic factor. The practical use of enzymes often requires elevated temperatures to increase productivity, prevent microbial contamination, improve the solubility of substrates and reduce the viscosity of the reaction medium. On the other hand, the stability of enzymes is affected by conditions such as heat, contact with chemicals and organic solvents, all of which cause denaturation. Amongst these, heat is by far the most important factor for the loss of the biological activity of enzymes and some correlation exists between thermal stability and other kinds of stabilization such as resistance to proteolysis. Thermal inactivation of enzymes is initiated by the partial reversible unfolding of their native structure which is followed by irreversible configurational/conformational changes. Processes such as aggregation, formation of "scrambled structures", cleavage of disulfide bridges, peptide bond hydrolysis, racemization of amino acid residues, deamidation, dissociation of prosthetic groups, isopeptide bond formation and oxidation of thiol/indole groups have been implicated during heat mediated denaturing of enzymes.
Enhancement of the thermal stability could alleviate most, if not all, of the problems associated with the use of native enzymes for various applications. Thermostabilization strategies followed during the past three decades consist of (i) addition of substances, (ii) chemical modification, (iii) cross-linking, (iv) use of anhydrous solvents (non-aqueous media), (v) protein engineering and (vi) immobilization. Of these, the immobilization technique is the most extensively used one for imparting thermal stability to enzymes. Enzymes immobilized on suitable substrates possess considerable advantages over those used in the soluble phase. They often show marked increase in stability and may be used in bioreactors for continuous processing, thereby cutting down on the costs in comparison with reactors utilizing these biocatalysts in solution. For example, using immobilized aminoacylase, the cost of amino acid production is reduced by 40% as against the soluble enzyme. In addition, immobilized biocatalysts are easily removable from reaction mixtures and have enhanced shelf life.
By definition, an immobilized enzyme is a protein physically localized in a certain region of space or converted from a water-soluble mobile state to a water-insoluble immobile condition. Protocols used for immobilizing enzymes can be categorized according to whether the protein becomes immobile by chemical binding or by physical retention. These consist of (i) binding of enzyme molecules to carriers through covalent bonds, (ii) by adsorptive interactions (physisorption), (iii) entrapment into gels, beads or fibres, (iv) cross-linking or co-crosslinking with bifunctional reagents and (v) encapsulation in microcapsules or membranes. Of these, the adsorptive procedures have become more or less obsolete due to the fact that the surfaces produced are too unstable to withstand mechanical stresses and chemical treatments involved in industrial processes. Immobilization through cross-linking has met with limited success because of the large amounts of enzyme required, the uncontrollable nature of the reaction which may lead to inactivation and the unsuitable mechanical properties of the resulting surfaces. The main disadvantages of the microencapsulation technique are that the molecular weight of the substrate has to be very low to allow diffusion across the membranous barrier and the capsules are very prone to enzyme leakage as they are relatively fragile. Furthermore, the polysaccharide-based polymeric materials used for entrapping enzymes into gels or beads suffer from the fact that strict sterile operating conditions must be maintained to prevent the growth of bacteria and fungii. With acrylamide monomers used for entrapment purposes, the conditions of photopolymerization may generate localized temperatures up to 60.degree. C. causing denaturing of the enzyme. With other polymeric systems, problems of enzyme loading, viability and stability have to be overcome for industrial applications.
Several reviews have appeared in the scientific and patent literature on the available choices of substrates and the protocols for covalently binding enzymes on them. The substrates in vogue range from inorganic materials such as porous glass, ceramics, silica and metal/metal oxides to organic materials such as the natural polymers cellulose, chitin and agarose and synthetic products like acrylates, polyamides, derivatized polystyrene and redox systems like polypyrrole. Biomolecules like the avidin-biotin system or bovine serum albumin are also being utilized. However, because of the problems of microbial growth on organic supports, and the consequent loss of activity, collapse of the structure and product contamination, there has been an increasing interest in the use of inorganic support materials, especially silica, controlled pore glass and ceramics.
The two factors to be considered in the selection of a method for the covalent linkage of an enzyme to a support are: the type of functional groups on the protein through which binding to the support is to be accomplished (and consequently the type of chemical reactions to the employed) and the physical/chemical characteristics of the support material with appropriate reactive functionalities grafted onto their surface. The functional groups on the enzymes which are available for covalent bonding are (1) amino (eta-amino groups of lysine and arginine and the N-terminal amino moieties of the polypeptide chains), (2) carboxyl groups of aspartic and glutamic acid and the C-terminal moieties, (3) phenol rings of tyrosine, (4) sulfhydryl groups of cysteine, (5) hydroxyls of serine, threonine and tyrosine, (6) the imidazole groups of histidine and (7) the indole groups of tryptophan. In practice, most of the covalent coupling reactions involve the amino, carboxy and mercapto moieties on the amino acids in the protein structure. The solid supports, in turn, must carry functional groups such as carboxyl, amino, formyl, epoxy, halo (chloro or bromo) and hydroxyl. A majority of solid supports either carry hydroxyls on their surfaces or can be easily modified by chemical or electrochemical means to introduce such hydroxylic groups.
Chemical reactions most commonly used for the interaction of the functionalities in the enzyme with those on the support materials consist of (1) the nucleophilic displacement of the surface hydroxyls on the supports activated with a sulphonyl chloride, 2-fluoro pyridinium tosylate or cyanuric chloride by the amino group on the protein, (2) nucleophilic addition of the protein amino group to a surface hydroxyl on the support which is activated with cyanogen bromide or carbonyldiimidazole or a chloroformate; or an analogous nucleophilic addition of the protein amino group to a carboxyl on the support surface which is activated as its N-hydroxysuccinimide ester, azide or with a diimide, (3) electrophilic addition of a diazonium functionality formed from an aromatic amino moiety on the support to the tyrosine residues on the enzyme, (4) electrophilic addition of the mercapto group on the cysteine moiety of the enzyme to a maleimide function introduced onto the surface of the support, and (5) cross-linking a surface amino group on the support to an amino group on the enzyme with a bifunctional reagent such as glutaraldehyde.
The thermal stability of enzymes covalently attached to support materials is significantly enhanced in comparison with the native enzyme. For example, Hayashi et al. (J. Appl. Polym. Sci. 1992, 44, 143) have observed that papain immobilized on polymethyl L-glutamate exhibited an activity up to three times higher than the native enzyme when maintained at 70.degree. C. in buffer solution for one hour. The free papain loses 90% of its initial activity at 75.degree. C. within 45 minutes. Raghunath and coworkers (Biotechnol. Bioeng. 1984, 26, 104) have demonstrated that urease immobilized on collagen-poly(glycidyl methacrylate) graft copolymer support was thermally stable up to 70.degree. C. and 40 days when stored at 4.degree. C. in a buffer solution. Davidenko et al. (Chem. Abstr. 1985, 102, 127894) have reported that urease adsorbed on carbon fibres is stable up to 65.degree. C. and retained 90% of its activity when stored for a month at 4.degree.-5.degree. C. Thermal stabilization up to 70.degree. C. in buffer solutions was also reported for chymotripsin by multi-point covalent attachment to aldehyde-agarose gels (Guisen et al. Biotechnol. Bioeng. 1991, 38, 1144) and for glucoamylase on periodate oxidized dextran (Lenders and Chricton, Biotechnol. Bioeng. 1988, 31,267). Asakura et al. (Polym.-Plast. Technol. Eng. 1989, 28, 453) immobilized alkaline phosphatase on Bombyx mori silk fibroin by cyanogen bromide and diazo coupling methods and have shown that while the free enzyme was totally deactivated at 65.degree. C., the enzyme coupled by the diazonium procedure retained 30% of its activity, in comparison with 10% for the cyanogen bromide-modified product. Yabushita and coworkers (Chem. Pharm. Bull. 1988, 36, 954) have shown that urokinase immobilized on an ethylene-vinyl acetate copolymer matrix retained more than 50% of its initial activity when kept for 8 hours at 45.degree. C., while the soluble enzyme lost almost all of its activity in 3 hours.
Margolin and coworkers (Eur. J. Biochem. 1985, 146, 625) effected a comparative evaluation of the stability and activity of enzymes immobilized on water-soluble and water-insoluble supports. Employing poly (N-ethyl-4-vinyl pyridinium bromide) (a polycationic support) and poly (methylacrylic acid) (a polyanionic support) for immobilizing a series of enzymes, these authors showed that pronounced thermal stabilization of penicillin amidase and urease could be achieved only if these enzymes are on the precipitated supports (in the insoluble form) and covalently attached to the polyelectrolyte nucleus. Thus, the thermal stability of polyelectrolyte complex-bound penicillin amidase increased seven-fold at pH 5.7, 60.degree. C. and three hundred-fold at pH 3.1, 25.degree. C., compared to the native enzyme. For urease, the thermal stabilization increases twenty-fold at pH 5, 70.degree. C.
The role of phospholipids as protective agents for maintaining the activity of antibodies, enzymes and receptors is well-documented. There is considerable evidence concerning the requirement of a lipid environment for sustaining the activity of enzymes. For example, it has been shown that a lipid-modified glucose oxidase enzyme electrode offers greater selectivity and stability for the analysis of glucose. Phospholipids may act as modulators of enzymatic reactions in addition to their role as obligatory cofactors for some membrane enzymes. Thus, it was shown (Niedzwiecka et al., Acta Biochim. Biophys. Hung., 1990, 25,47), that the purified lymphocyte 5'-nucleotidase reconstituted into lipid bilayer demonstrates remarkable stability on storage at 4.degree. C. The liposome incorporated enzyme from chicken gizzard is five times more stable at 56.degree. C. than the enzyme in the detergent solution, indicating that the phospholipids play a role in preventing the denaturing process.
Rosenberg, Jones and Vadgama (Biochim. Biophys. Acta 1992, 1115, 157) encapsulated glucose oxidase in liposomes and found that electrodes coated with a nitro-cellulose membrane carrying these liposome-enzyme formulations exhibited extended linear range of response. The enzyme activity was found to be partially dictated by the liposomal bilayer permeability, and therefore, the enzyme affinity for its substrate could be regulated by using liposomes prepared from different lipids such as dimyristoyl, dipalmitoyl and distearoyl-phosphatidylcholine. It has also been shown by Kotowski and Tien (Bioelectrochem. Bioenerg. 1988, 19, 277) that glucose oxidase could be covalently immobilized on a polypyrrole-supported bilayer lipid membrane surface and the enzyme-substrate reaction could be followed by cyclic voltammetry. The phospholipid functions as an electric switch during this analysis, besides supplying the natural biomembrane-type environment to the enzyme.
Besides thermal inactivation, the extent of activity exhibited by an immobilized enzyme is also dependent upon aspects such as the chemical procedure used to effect immobilization, the spacer chain length and the pH of the buffering medium in which the enzyme-substrate reactions are carried out. For example, Comfort et al. (Biotechnol. Bioeng. 1988, 32, 554) evaluated the immobilization yields of heparinase and bilurubin oxidase on agarose and acrylic beads activated by four different reagents, viz. cyanogen bromide, carbonyldiimidazole, oxirane and tresyl chloride, respectively. They found that while heparinase was bound in 90% yield (with 50% active enzyme) by the cyanogen bromide method, bilurubin oxidase was preferentially linked. (50-55% maximum yield, with 25-30% active enzyme) by the tresyl chloride and oxirane displacement. However, in both cases, nearly 40-50% of the immobilized enzymes were leached out when allowed to stand in buffer for a short time. Przybyt and Sugier (Anal. Chim. Acta 1990, 239, 269) investigated the activity of urease immobilized on oxidized tungsten electrodes by electrochemistry. The covalent binding protocol followed by these authors consisted of initially silanizing the metal oxide surface with gamma-aminopropyltriethoxysilane and then cross-linking the enzyme with either cyanuric chloride or hexamethylene diisocyanate or glutaraldehyde. They found that the lifetime of the enzyme electrodes with the cyanuric chloride linker was only one day. In comparison, the lifetimes of electrodes prepared by employing glutaraldehyde and the diisocyanate cross-linkers were 29 and 22 days, respectively. The life-time of the enzyme electrode, obtained by the direct cross-linking of the metal oxide surface with the enzyme through hexamethylene diisocyanate (without prior silanization) was 19 days. Furthermore, these authors noted profound effects on the electrode response due to factors such a the nature of the buffer, its concentration and ionic strength.
The importance of the spacer chain length towards the retention of the activity of an immobilized enzyme on a given surface has been demonstrated by several groups of workers. For instance, Kennedy and Cabral (in Methods in Enzymology, Vol. 135, pp. 117-130, Academic Press, San Diego, 1987) examined the linking of glucoamylase to control pore glass activated with titanium tetrachloride. The substrates were initially treated with ammonia (no carbon spacer), 1,2-diaminoethane (a two-carbon spacer) and hexamethylene diamine (a 6-carbon spacer) and then cross-linked with the enzyme through glutaraldehyde. The six carbon spacer-carrying substrate exhibited an activity retention of 12% relative to the activity of the soluble enzyme, while the figures were 1.5% and 3.2% for the no carbon and two carbon spacer, respectively. Jayakumari and Pillai (J. Appl. Polym. Sci. 1991, 42, 583) observed that the direct coupling of papain to carboxylated polystyrene yielded only 5% active enzyme, while binding of the same enzyme to the same support through glutaric anhydride cross-linker produced 30% of active enzyme. However, the maximum activity retention (54%) was obtained when papain was linked to hydroxymethyl polystyrene through polyethylene glycol (PEG 600) cross-linker. These authors also demonstrated that increasing cross-link densities decreased the total immobilization yields as well as the amount of active enzyme. Furthermore, rigid supports lowered total/active enzyme yields in comparison with flexible supports. Schuhmann et al. (J. Amer. Chem. Soc. 1991, 113, 1394) showed that the electrical communication between the redox centres of glucose oxidase and vitreous carbon electrodes is more effective when a long chain diamine was used to cross-link the aldehyde functionalities of ferrocene and those of glucose oxidase obtained by the oxidation with periodate. Reduction of electron-transfer distances between the redox centre of the enzyme and the peripherally bound ferrocene relay and between the relay and the electrode due to penetration of the relay to a sufficient depth by the enzyme was postulated to be responsible for their observations. Kobayashi et al. (J. Colloid Interface Sci. 1991, 141, 505) have reacted microfine magnetic particles of magnetite with APTES and then cross-linked the surface with a protease; thermolysin, with glutaraldehyde. They also utilized omega-aminohexylaminopropyltrimethoxysilane, 4-aminobutylaminopropyltrimethoxysilane and 2-aminoethyl-aminopropyltrimethoxysilane and showed that maximum enzymatic activity was exhibited by the hexyl-silane (50% higher than with APTES).
The report of Williamson et al. (Anal. Letters 1989, 22, 803), however, contradicts the above findings on the spacer length, when an antibody, rather than an enzyme, is immobilized to a support. These authors covalently attached anti-T.sub.2 mycotoxin monoclonal antibodies on quartz fibres by three techniques. The first two consist of the activation of the surface hydroxyls of quartz with p-toluene sulphonyl chloride or p-nitrophenylchloroformate, followed by the direct attachment of the antibody. The third method involves initial silanization of quartz with APTES followed by cross-linking of the antibody with glutaraldehyde. Almost the same amount of activity was found to be exhibited by the antibody on all of the above three surfaces. However, the thermal stability of the antibody on the APTES-modified surface at 50.degree. C. was considerably better than the antibody surfaces prepared with the other two reagents. Significantly, treatment of the sulphonyl chloride or chloroformate activated quartz with hexamethylene diamine, prior to the immobilization of the antibody with glutaraldehyde, did not improve the activity of the bound antibody, in spite of the six-carbon spacer.
The above brief summary of the thermal and a thermal factors responsible for the deactivation of enzymes indicates that even immobilized enzymes are not stable above 60.degree.-70.degree. C. In a number of instances, nearly 50% of the immobilized enzyme is leached out by washing with a buffer or detergent. Use of cross-linkers during the immobilization of the enzymes also has a detrimental effect on the retention of the activity by the immobilized biomolecules. Recent advances in the isolation of thermostable enzymes utilize thermophilic bacteria and considerable thermal stability has been claimed for the enzymes made by this route. However, a recent report by Brosnan and coworkers (Eur. J. Biochem. 1992, 203, 225) demonstrates that alpha-amylase isolated from Bacillus stearothermophilus is irreversibly deactivated at 90.degree. C. in 1.9 minutes at pH 5.0.
Although a large number of publications in documented literature have clearly indicated that phospholipids exert a stabilizing effect on the activity of enzymes, enzyme preparations so far known have only utilized encapsulations in phospholipid liposomes. In two earlier patents (U.S. Pat. No. 4,824,529 {1989] and U.S. Pat. No. 4,637,861 [1987]), as well as in a recent publication (Anal. Chim. Acta 1989, 225, 369), we have demonstrated that phospholipids can be covalently attached to different kinds of supports. As analogues of natural biomembranes, these phospholipids are expected to impart greater stability than hitherto known to enzymes, if the two bio-entities could be covalently linked. It is envisaged that the combination of a suitable spacer chain and immobilization to a support through a phospholipid would enable the formation of thermally very stable enzyme systems with extended operational and storage stabilities in the solid state (without any buffers), for a variety of applications.
It is therefore an object of the present invention to provide new compounds suitable as spacers as well as linkers for the covalent immobilization of enzymes and other biologically active substances either directly or through an intermediate compound, onto a substrate.
It is a further object of the present invention to provide new phospholipids suitable for covalent binding to the substrate through a spacer compound and to the bioactive molecule.
It is another object of the present invention to provide preparations comprising immobilized biologically active substances, e.g. enzymes, bound to the substrate through the spacer compounds and optionally also through the phospholipids.
It is still another object of the present invention to provide methods for the preparation of the spacers and phospholipids utilized in the present invention.